Effect of different biochar additions on the change of carbon nitrogen content and bacterial community in meadow soils

ABSTRACT

Meadow soils are one of the important agricultural soil types, biochar is a good soil amendment, whereas the studies on the effects of biochar additions on carbon and nitrogen contents, as well as bacterial communities of meadow soils, were seldom carried out. The effects of one nitrogen addition rates (no addition, 0.2% addition) and two biochar addition patterns (no addition, 1% addition, 3% addition) on the carbon and nitrogen contents, as well as bacterial communities of meadow soils, were investigated by conducting a one-year indoor incubation experiment. Biochar addition increased the relative abundance of Proteobacteria, Chloroflexi and Bacteroidota.   The combination of biochar and nitrogen source significantly increased the total organic carbon, ammonium nitrogen, nitrate nitrogen and total nitrogen content of the meadow soil. Therefore, the addition of moderate amounts of biochar to meadow soils can contribute to sustainable ecological agriculture as a form of soil improvement.

KEYWORDS:

• Biochar
• meadow soil
• total organic carbon
• total nitrogen
• bacterial community
• functional genes analysis

1 Introduction

Meadow soils are fertile soils with high organic matter content, thick humus layer, good soil mass structure, rich in nutrients and sufficient water [1]. Meadow soils are usually located in low-lying plains and are susceptible to flooding and nutrient loss. Years of tillage and excessive addition of chemical fertilizers also lead to soil consolidation, as well as decrease in organic carbon content, nutrient content, water permeability and storage capacity, seriously constricting on the sustainable development of green agriculture. Research on the application of biochar for soil improvement had focused on black, loess and loam soils; however, the research on the improvement of meadow soils is lacking [2–4].

Biochar is a char material obtained by pyrolysis of biomass under anaerobic and higher temperature conditions [5,6]. Biochar has a high pH value, large specific surface area, rich functional groups, high cation exchange capacity and great potential to improve soil quality [7]. The biochar prepared from agricultural wastes such as straw or pig manure returning into the field can not only realize the resource utilization of wastes but also effectively improve soil fertility [8,9]. It has been suggested that biochar addition can increase the yield of crops such as legumes and corn [10]. Previous studies had focused on the improvement of black soils by biochar [11,12]; however, the effect of biochar addition on meadow soils had rarely been reported.

Biochar has a special pore structure and huge specific surface area and is rich in nutrient elements. At the same time, the addition of biochar increases the content of soluble organic and inorganic carbon in the soil in the short term, which affects the soil bacterial community [13,14]. Biochar-driven priming effects can have positive or negative effects on soil organic carbon mineralization. Total soil organic carbon is one of the important indicators of soil fertility. Moreover, the addition of biochar can effectively increase total soil organic carbon and inhibit organic carbon mineralization [15]. There are also former studies showing that biochar addition promotes the mineralization of organic matter, which conversely reduces soil total organic carbon [16,17]. Biochar made from straw has a high ratio of carbon to nitrogen, and biochar addition introduces carbon as well as nitrogen sources; therefore, biochar would affect the total nitrogen, ammonia nitrogen and nitrate nitrogen content of the soil [18]. However, the effect of biochar-induced initiation effects (microbial mechanisms) on the different forms of carbon and nitrogen content of meadow soils is unclear. Considering the actual agricultural production activities, the addition of nitrogen source is essential. Thus, it is also necessary to investigate the effect of biochar when adding nitrogen source.

Soil bacterial communities are important parts of the soil ecology [19,20], and biochar addition will significantly affect black or loess soil bacterial communities [21,22]. However, the mechanism of biochar addition affecting the changes of the bacterial community, carbon and nitrogen contents in meadow soils was not clear. The particle size of biochar has a large influence on its performances, such as water retention capacity and adsorption capacity of ammonia nitrogen [23]. Moreover, this factor is likely to have an impact on the relationship between the action of soil microbial communities and carbon and nitrogen content in meadows. Nevertheless, in our literature research, few works had focused on this aspect. In this study, biochar of different particle sizes was also added to meadow soils.

The purpose of this study was to investigate the effects of biochar and nitrogen source addition on the carbon and nitrogen content as well as bacterial community of meadow soil. We found out that (1) in the case of biochar addition, nitrogen addition altered the bacterial community and significantly increased soil carbon and nitrogen content; (2) in the case of nitrogen addition, biochar addition altered the bacterial community and significantly increased soil carbon and nitrogen content, and small particle size biochar was more effective.

2 Materials and methods

2.1 Soils

Selected farmland soils around Harbin, Heilongjiang Province, China. The region has a mid-temperate continental monsoon climate. The average annual temperature and precipitation are 0–11°C mm and 550 mm, respectively. The soils belonged to meadow soils, according to the Chinese soil classification system test. The soils were collected from the farmland at 0 ~ 20 cm depth, and then straw and small stones were removed. At last, the soil was mixed well and passed through 10-mesh sieve. The physical and chemical properties of the soils we determined are shown in Table S1.

2.2 Biochar

We prepared biochar using maize straw collected from the farmlands around Harbin. First, we dried the straw at room temperature and then cut it into small pieces (<2 cm) and mixed well. At last, the straw was put in a special homemade machine (Figure S1) and pyrolyzed under anoxic conditions. The heating rate was 20°C/minute. The pyrolysis temperature was set at 400°C and maintained for 2 h. After the biochar was cooled, it was taken out and collected. The properties of biochar are shown in Table S2.

2.3 Experimental design

Eight treatments were designed in this study, including nitrogen (urea), raw biochar (0.18 mm ~ 2.00 mm) and small biochar (<0.18 mm) addition. Blank group (B1), 1% biochar treatment (B2), 3% biochar treatment (B3), nitrogen treatment (B4), 1% biochar and nitrogen treatment (B5), 3% biochar and nitrogen treatment (B6), 1% small particle size biochar and nitrogen treatment (B7) and 3% small particle size biochar and nitrogen treatment (B8). Specific treatment conditions are presented in Table S3. The effect of bacterial community chang in meadow soil was slow and long-lasting. In order to study the change of bacterial community and its effect on the carbon and nitrogen content of meadow soil after adding biochar in this study, we set up five experimental cycles. The experiment lasted from June 2021 to June 2022, each trial cycle lasted 2 months, for a total of 1 year.

Each treatment was carried out in a polyethylene dome-shaped cassette (0.5 kg soil) with an appropriate number of small holes on the top lid. The biochar, urea and soil were mixed uniformly, and the mass was weighed every 5 days to replenish the amount of water. Five batches of independent destructive sampling were conducted, only one batch of sampling was conducted for each test cassette, and the samples were not put back to the original place after sampling. Total organic carbon, soluble organic carbon, soluble inorganic carbon, microbial biomass carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen, microbial biomass nitrogen and bacterial community were determined by sampling appropriate amount of soil. Three replications were performed for each treatment.

2.4 Experimental methods

A pH meter (PHS-3 G, China) was used to measure the pH of the soil at a soil/water ratio of 1:5. The soil bulk and water content were determined using a ring knife. Determined their particle size by laser particle size distribution (Mastersizer 2000, UK). Total organic carbon of soil was determined by potassium dichromate oxidation spectrophotometric method using the UV spectrophotometer (T6 New Century, China) [24]. Soil ammonia nitrogen was determined by Nessler’s reagent spectrophotometry method [25]. Soil nitrate nitrogen was determined by ultraviolet spectrophotometry method [26]. We used the alkaline potassium persulfate digestion method to measure the total nitrogen content [27].

This experiment used high-throughput sequencing to measure the soil bacterial microflora. Soil (0.25–0.5 g) was added into a 2 mL centrifuge tube, and then 500 µL of buffer and 0.25 g of grinding beads were added. The tube was shaken for 15 min until the sample was homogeneously mixed using a TGrinder H24 tissue grinding homogenizer (OSE-TH-01) (shake for 30 s at 30 s intervals for 2 cycles). Centrifugation was done at 12,000 rpm for 1 min, and the supernatant (~500 µL) was transferred to a 2 mL centrifuge tube. Nucleic acid was extracted using the Soil Genomic DNA Extraction Kit TGuide S96 Magnetic Bead Method. PCR modified primers 341F (5′ -CCT AYG GGR BGC ASC AG-3′) and 806R (5′ -GGA CTA CNN GGG TAT CTA AT-3′) were used to amplify the 16S rRNA gene from bacteria DNA [28]. PCR kit (Hongyue Technology Co., China) was stored at −20°C before being analyzed. The PCR products were sequenced by biomaker limited company (Beijing, China). USEARCH (version 10.0) at a 97% similarity level was used to cluster sequences. The created library was first subjected to library quality control, and qualified library was sequenced by Illumina Novaseq 6000. Raw image data files obtained were converted into sequencing reads for base call analysis from high-throughput sequencing (Illumina Novaseq), and the FASTQ (short for fq) file format was stored as the results. In order to filter OTUs, all sequences sequenced had defaulted threshold of 0.005% [29]. Denoised QC data by using the DADA2 method in QIIME2 (version 2020.6) [30].

2.5 Data analysis

The carbon content of biochar is 73.19% (Table S2), so by adding 1% biochar, the theoretical total soil organic carbon content should be increased by 7.32 g/kg; urea has 20% carbon content, and by adding 0.2 g of urea to 0.5 kg of soil, the theoretical total soil organic carbon content should be increased by 0.08 g/kg, and the two of them should be added together to increase the theoretical total soil organic carbon content by 7.40 g/kg. All the experiments were conducted in triplicate. Data were statistically and analytically conducted by Origin 2021 and SPSS 26. Complements the theoretical increase in how to calculate total soil organic carbon content. Analysis of variance (ANOVA) was conducted, followed by the least significant difference (LSD) test with biochar addition as the primary factor at a significance level of P < 0.05 for the following variables. Cannon 5.0 analyzed the relationship between soil properties and soil bacterial community composition using redundancy analysis (RDA). Linear discriminant analysis effect size (Lefse) analysis was performed on intergroup samples. The PICRUSt2 software was used to annotate species with the characteristic sequences to be predicted in the phylogenetic tree available in the software, and the IMG microbial genomic data was used to export functional information and thus to infer the functional gene composition of the samples. The functional differences were analyzed between eight samples, including Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups of proteins (COG). The FAPROTA database was used for functional annotation and prediction of microorganisms and analyzed by drawing with the STAMP software. Data are presented as means with standard and deviations.

3 Results

3.1 Biochar addition effects the carbon content of meadow soils

As shown in Figure 1a, compared with treatment B1 (without biochar), the total organic carbon content of meadow soil in treatments of B2 (with 1% biochar) and B3 (with 3% biochar) increased by 32.37% and 63.11%, respectively, therefore, biochar addition significantly increased the total organic carbon content of meadow soils. Compared with treatment B4 (urea addition), the total organic carbon content of meadow soil in treatments of B5 (urea and 1% biochar addition) and B6 (urea and 3% biochar addition) increased by 35.06% and 85.28%, respectively. The total organic carbon content of meadow soils in treatments of B7 (with 1% biochar) and B8 (with 3% biochar) increased by 54.98% and 100.26%, respectively. Compared to the first cycle at the beginning of the experiment, the total organic carbon content in the eight treatments at the fifth cycle increased by 11.90%, 28.52%, 41.77%, −0.05%, 15.90%, 26.15%, 15.90% and −0.04%, respectively, as shown in Figure 1b. In the first four cycles of the experiment, soluble total organic carbon increased with the biochar addition, while decreased in the fifth cycles. When 1% biochar was added, the theoretical increase in total soil organic carbon content should be 7.31 g/kg, while the actual increase in soil organic carbon content was 7.9 g/kg. When 1% biochar and urea were added, the theoretical increase in total soil organic carbon content should be 7.40 g/kg, while the actual increase in soil organic carbon content was 8.1 g/kg.

Figure 1. Effect of biochar addition on carbon content changes in meadow soils. Total organic carbon (a); soluble organic carbon (b); soluble inorganic carbon (c); microbial biomass carbon (d). Different letters show the significant difference according to the LSD test at 5% probability level (P < 0.05).

Soluble inorganic carbon in soil mainly includes carbonate and bicarbonate (shown in Figure 1c). The soluble inorganic carbon content was relatively low in all treatments at the beginning of the experiment. However, the biochar significantly increased the soluble inorganic carbon content of meadow soils. In the fifth cycle of the experiment, the soluble inorganic carbon content in treatments of B1, B2 and B3 were 212.74 mg/L, 301.98 mg/L and 295.48 mg/L, respectively; moreover, the difference between treatment of 1% biochar and 3% biochar addition was insignificant. When nitrogen source was added, the difference of soluble inorganic carbon content between 1% biochar and no biochar was insignificant; however, 3% biochar significantly increased the soluble inorganic carbon content of meadow soils.

In the fifth cycle of the experiment, biochar increased soil microbial biomass carbon content without adding nitrogen source. However, when nitrogen source addition was added, the microbial mass carbon in 3% biochar treatment was lower than that of 1% biochar treatment (Figure 1d). The microbial carbon content increased and then decreased with biochar addition increased, which may be due to excessive addition of biochar resulting in an excessive increase in ammonia nitrogen content, which was toxic to some microorganisms.

3.2 Biochar addition effects nitrogen content of pain meadow soils

The overall total nitrogen content of meadow soils slightly increased as the experiment progressed (Figure 2a). In the last three cycles of the experiment, the total nitrogen content significantly increased in meadow soil. Compared to the first cycle, the total nitrogen content in the eight treatments at the fifth cycle increased by 50.70%, 51.23%, 59.74%, 42.78%, 44.50%, 45.77%, 41.79% and 33.20%, respectively, the increase in total nitrogen content was significant. The more biochar added, the more total nitrogen increased at the end of the experiment compared to the beginning of the experiment. The enhancement of soil total nitrogen content was 0.32 g/kg, 0.36 g/kg and 0.80 g/kg when the soil was added by 3% biochar, 3% biochar and urea and 3% small particle size biochar and urea.

Figure 2. Effect of biochar addition on nitrogen content changes in meadow soils. Total nitrogen (a); ammonia nitrogen (b); nitrate nitrogen (c); microbial biomass nitrogen (d). Different letters show the significant difference according to the LSD test at 5% probability level (P < 0.05).

In the first cycle of the experiment, biochar increased the ammonia nitrogen content of meadow soils (Figure 2b), while nitrogen source significantly increased the ammonia nitrogen content of meadow soils, with the lowest ammonia nitrogen content of 15.61 mg/L in the treatment of B1 and the highest ammonia nitrogen content of 45.45 mg/L in the treatment of B8. In the fifth cycle of the experiment, the addition of biochar still increased the ammonia nitrogen content of meadow soils.

Overall, the nitrate nitrogen content of the meadow soil increased at the beginning of the test and then gradually decreased (Figure 2c). In the fifth cycle of the experiment, the nitrate-nitrogen content in treatments of B1, B2 and B3 were 7.77, 10.13 and 16.27 mg/L, respectively; moreover, the nitrate-nitrogen content in treatment of B4, B5 and B6 were 41.77, 42.88 and 44.80 mg/L, respectively; therefore, biochar increased the nitrate nitrogen content of meadow soils. In the fifth cycle of the experiment, biochar increased the microbial biomass nitrogen in the soil without the addition of a nitrogen source, while it was opposite when a nitrogen source was added (Figure 2d).

3.3 Effect of biochar addition on meadow soil microbial diversity indices

Table 1 shows that single 1% biochar increased ACE, Chao1, Simpson and Shannon indices, while 3% biochar addition decreased ACE, Chao1 and Shannon indices. Under the condition of nitrogen addition, the ACE index and Chao1 index were reduced, indicating that biochar addition reduced the abundance of bacterial community after 1 year of the experiment. Biochar reduced both Simpson index and Shannon index, moreover, biochar reduced the abundance and evenness of bacterial communities. For treatments of B7 and B8 with the addition of small particle size biochar, Simpson index and Shannon index were smaller than those for treatments of B5 and B6, indicating that the effect of adding small particle size biochar reduced the abundance and evenness of bacterial communities was more obvious than that of adding biochar.

Table 1. Meadow soils microbial alpha diversity.

Sample	Feature	ACE	Chao1	Simpson	Shannon	PD-whole-tree	Coverage
B1	908	909.79	910.33	0.997	9.16	43.08	0.999
B2	924	924.89	926.00	0.997	9.21	44.12	0.999
B3	892	892.55	898.00	0.997	9.16	44.37	0.999
B4	894	894.86	904.00	0.997	9.08	44.15	0.999
B5	862	862.44	863.00	0.996	8.98	45.96	0.999
B6	827	827.34	827.25	0.997	8.99	41.42	1.0
B7	856	856.67	857.20	0.997	9.00	41.14	0.999
B8	754	754.00	754.00	0.995	8.67	4.31	1.0

3.4 Species abundance clustering heat map

The result of the clustering heat map is shown in Figure 3. Horizontally, the eight treatments were clustered into four categories: B1, B2 and B3 were the first category, B4 and B5 were the second category, B6 and B7 were the third category, and B8 was the last category. Longitudinally, the eight samples were classified into six categories at the phylum level, with Myxococcota being the most similar to Methymoirabilota.

Figure 3. Heat map of meadow soils bacterial communities.

3.5 Effect of biochar addition on meadow soils microbial community structure

As shown in Figure 4a, Proteobacteria, Acidobacteriota, Actinobacteriota, Gemmatimonadota, Chloroflexi, unclassified Bacteria, Bacteroidota, Myxococcota, Nitrospirota and Methylomirabilota were the top 10 bacteria in terms of abundance at the phylum level. As for treatments of B1, B2 and B3, biochar addition increased the relative abundance of Proteobacteria, Chloroflexi and Bacteroidota while decreased the relative abundance of Actinobacteriota and Gemmatimonadota. As for treatments of B4, B5, B6, B7 and B8, biochar addition decreased the relative abundance of Myxococcota and Nitrospirota. Under the same biochar addition, the nitrogen source addition increased the relative abundance of Nitrospirota; however, it decreased with biochar content.

Figure 4. Relative abundance of meadow soil bacterial communities. Phylum level (a); class level (b); order level (c).

As shown in Figure 4b, Gammaproteobacteria, Alphaproteobacteria, Vicinamibacteria, Gemmatimonadetes, Actinobacteria, Acidimicrobiia, Bacteroidia Blastocatellia and Nitrospiria were the top nine bacteria in terms of abundance at the class level. For treatments of B1, B2 and B3 biochar increased the relative abundance of Alphaproteobacteria, Acidimicrobiia, Bacteroidia and Blastocatellia while decreased the relative abundance of Vicinamibacteria, and Gemmatimonadetes. For treatments of B4, B5, B6, B7 and B8, biochar decreased the relative abundance of Nitrospiria, and the lowest relative abundance of Nitrospiria was only 1.25% in treatment of B8.

As shown in Figure 4c, Vicinamibacterales, Sphingomonadales, Burkholderiales, Gemmatimonadales, unclassified_Bacteria, Xanthomonadales, Rhizobiales, PLTA13, Nitrospirales and Microtrichales were the top 10 bacteria in terms of relative abundance at the order level. For treatments of B1, B2 and B3, biochar increased the relative abundance of Rhizobiales, while decreased the relative abundance of Gemmatimonadales. For treatments of B4, B5, B6, B7 and B8, biochar decreased the relative abundance of PLTA13, Nitrospirales.

3.6 Effect of biochar on the relative abundance of nitrifying bacteria and nitrogen fixation bacteria in meadow soils

As shown in Figure 5a, without nitrogen addition, biochar addition (treatments of B1, B2 and B3) increased the relative abundance of Nitrosomonadaceae and Nitrospira in meadow soils; however, under the condition of nitrogen added, biochar addition (treatments of B4, B5, B6, B7 and B8) decreased the relative abundance of nitrifying bacterial populations in meadow soils.

Figure 5. Effect of biochar addition on the relative abundance of nitrogen fixation and nitrifying bacterial populations. Nitrifying bacteria (a); nitrogen fixation (b).

As shown in Figure 5b, biochar increased the cumulative relative abundance of bacterial communities with nitrogen fixation effect, and the highest relative abundance was 17.13% in treatment B8.

3.7 Analysis of significance of differences between groups (LEfSe analysis)

As shown in Figure 6a, the biochar addition significantly increased the relative abundance of Rhizobiaceae, Lysobacter and Xanthomonadales, while decreased the relative abundance of Cellulomonadaceae and Pedosphaerales. Both Rhizobiaceae and Xanthomonadales are aerobic bacteria and biochar addition improved the permeability of soil, thus increasing their relative abundance.

Figure 6. Effects of biochar and nitrogen addition between groups differences in bacterial communities of meadow soils. Biochar differentiation (a); nitrogen differentiation (b).

As shown in Figure 6b, the nitrogen addition significantly increased the relative abundance of Actinobacteriota and Gammaproteobacteria, while decreased the relative abundance of Acidobacteriota and Elusimicrobiota.

3.8 Redundancy analysis (RDA)

As shown in Figure 7, the smaller angle between Myxococcota and Methylomirabilota as well as the longer length of the arrow indicated a good correlation between them and a positive correlation with microbial load carbon content. Actinobacteriota, Bacteroidota and Chloroflexi were positively correlated with total organic carbon, microbial mass nitrogen, ammonia nitrogen and total nitrogen content. The analysis showed that the first axis and the second axis explained 77.55% and 20.23% of the information. This indicated that total nitrogen and total organic carbon content are the main soil environmental factors affecting the structure of soil bacterial communities at the phylum level.

Figure 7. Meadow soil bacterial communities and nature of RDA in different experimental groups. NH₄+-N: ammonia nitrogen; NO₃⁻-N: nitrate nitrogen; MBC: microbial biomass carbon; MBN: microbial biomass carbon; SOC: soil organic carbon; TN: total nitrogen.

3.9 Functional gene prediction analysis

The analysis of the composition and differences of KEGG metabolic pathways can be used to observe the differences and changes in the metabolic pathways of functional genes in microbial communities between samples in different treatments, which is an effective means to study the changes in metabolic functions that occur in community samples to be adapted to environmental changes, the following are all described in descending order of gene difference size. The analysis results between treatments of B1 and B3 are shown in Figure S2. A total of 6 categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Membrane transport, Cellular community – prokaryotes, Cell motility and decreased the expression of genes such as Global and overview maps, Carbohydrate. The analysis results between treatments of B4 and B6 are shown in Figure S3. A total of 20 categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Carbohydrate metabolism, Xenobiotics biodegradation and metabolism, Amino acid metabolism and so on, decreased the expression of genes such as Translation, Global and overview maps, Cell motility and so on. The analysis results between treatments of B4 and B8 are shown in Figure S4. A total of 23 categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Membrane transport, Xenobiotics biodegradation and metabolism, Amino acid metabolism and so on, decreased the expression of genes such as Global and overview maps, Translation, Glycan biosynthesis and metabolism and so on.

Clusters of Orthologous Groups of proteins (COG) is a commonly used protein functional classification database for prokaryotes, the following are all described in descending order of difference. The analysis results between treatments of B1 and B3 are shown in Figure S5. A total of 10 categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Inorganic ion transport and metabolism, Posttranslational modification and so on, decreased the expression of genes such as Carbohydrate transport and metabolism, Transcription, Defense mechanisms and so on. The analysis results between treatments of B4 and B6 are shown in Figure S6. A total of 18 categories were counted under the second stratum with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Inorganic ion transport and metabolism, Lipid transport and metabolism, Secondary metabolites biosynthesis and so on, decreased the expression of genes such as Amino acid transport and metabolism, Defense mechanisms, Cell cycle control and so on. The analysis results between treatments of B4 and B8 are shown in Figure S7. A total of 20 categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Signal transduction mechanisms, Lipid transport and metabolism, Secondary metabolites biosynthesis and so on, decreased the expression of genes such as Cell wall/membrane/envelope biogenesis, Translation, ribosomal structure and biogenesis and so on.

The species abundance table entered by PICRUSt2 is currently only available for species annotation using green gene database, in which FAPROTAX is more flexible, as it identifies the genus and species names of bacteria. FAPROTAX is suitable for functional annotation prediction of biogeochemical cycling processes in environmental samples, the following were all described in descending order of difference. The analysis results between treatments of B1 and B3 are shown in Figure S8. A total of nine categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Nitrogen fixation, Aerobic Chemoheterotrophy, decreased the expression of genes such as Chitinolysis, Predatory or exoparasitic, Ureolysis and so on. The analysis results between treatments of B4 and B6 are shown in Figure S9. A total of 21 categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Fermentation, Chitinolysis, aerobic Chemoheterotrophy and so on, decreased the expression of genes such as Predatory or exoparasitic, Animal parasites or symbionts, Human pathogens all and so on. The analysis results between treatments of B4 and B8 are shown in Figure S10. A total of 22 categories were counted under the second stratum, with the proportion of genes differing by more than 0.02%. Biochar increased the expression of genes such as Aromatic compound degradation, Chemoheterotrophy, Chitinolysis and so on, decreased the expression of genes such as Predatory or exoparasitic, Animal parasites or symbionts, Human pathogens all, Nitrogen fixation and so on.

4 Discussion

4.1 Effects of biochar addition on the carbon content of meadow soils

In this study, a one-year cycle experiment was conducted. Adding biochar can achieve carbon sequestration by increasing hard-to-decompose organic carbon in soil [31]. When biochar was added to the soil, it releases dissolved organic carbon under physical, biological and chemical action [32]. Therefore, the initial biochar increased the dissolved organic carbon content of meadow soil. Dissolved organic carbon is highly degradable and has a very fast turnover rate, while biochar can reduce the conversion rate of total soil organic carbon to dissolved organic carbon by retaining humic acid in the soil and enhancing soil water retention [33], which may be the reason why the dissolved organic carbon content decreased with biochar addition at the fifth cycle of the experiment. Meanwhile, at the beginning of the experiment, biochar significantly increased the dissolved inorganic carbon content, and the larger specific surface area of biochar improved the permeability of the soil and adsorbed carbon dioxide and carbonate [34]. However, carbonate adsorbed by biochar can undergo hydrolysis reactions on its surface [35], which may be the reason that the dissolved carbonate content did not strictly increase with biochar in the late stage of the experiment. In this experiment, it was found that with the addition of nitrogen source, the total organic carbon content of meadow soils still increased after the addition of raw biochar compared with the first cycle and the fifth cycle of the experiment. Meanwhile, the enhancement of total organic carbon content of meadow soil was higher than the carbon content of biochar. This may be due to two factors: biochar did not decompose rapidly after addition, biochar changes the structure and functional genes of soil bacterial community, which reduced the mineralization of organic carbon and enhanced the carbon sequestration effect. When a small particle size biochar was added, the total organic carbon content was higher than that of raw biochar. This may be due to the greater effect of small particle size biochar on the soil bacterial community, which more obviously reduced the mineralization efficiency of organic carbon.

4.2 Effects of biochar addition on the nitrogen content of meadow soils

Biochar significantly increased the ammonia nitrogen content when adding urea due to the good adsorption effect of biochar on ammonia nitrogen [36]. It has been reported that biochar can enhance biological nitrogen fixation by altering the bacterial community [37], results in this study also showed that biochar increased the relative abundance of nitrogen-fixing bacteria and contributed to biological nitrogen fixation, which may be one of the reasons that biochar increased the ammonia nitrogen content of meadow soils. The initial adding of biochar increased the ammonia nitrogen content and the relative abundance of nitrifying bacteria [38], which promoted nitrification and produced more nitrate nitrogen. Meanwhile, biochar adsorbed ammonia nitrogen and also some nitrate nitrogen [39], leading to the increase in nitrate nitrogen content in the meadow soil. This is one of the reasons why biochar addition increased the total nitrogen content of meadow soils. At the same time, the combination of biochar and nitrogen fertilizer can inhibit soil organic nitrogen cycling and promote soil organic nitrogen accumulation [40], contributing to the accumulation of total nitrogen. Our experiment also found that small particle size biochar was more effective in enhancing total soil nitrogen. This may be due to the fact that the small particle size biochar improved the stability of soil aggregates, further enhancing the soil nitrogen fixation capacity [41]. It is also possible that smaller-sized biochar may have a greater surface than the larger-sized biochar, showing a greater biochar adsorption effect. Microbial biomass carbon and nitrogen are influenced by soil microbial community fluctuations, and the effect of biochar addition on microbial biomass carbon and nitrogen content is influenced by soil type and pH [42]. The soil microbial carbon and nitrogen content of soil microorganisms is higher when the soil quality is better and the pH is moderate. Biochar addition can reduce soil bulk weight, improve soil permeability and increase soil pH, so biochar addition has a more significant effect on the microbial carbon and nitrogen content of soils with higher bulk weight, poorer permeability and lower pH. Overall, biochar increasing the microbial carbon content was probably due to the fact that some microorganisms used the nutrients from biochar to promote their own life activities [43], while biochar increased the microbial carbon utilization efficiency (microbial growth to carbon uptake ratio) [44].

4.3 Effects of biochar addition on the bacterial communities of meadow soils

Soil bacterial communities have an important impact on soil ecology, and biochar addition had a significant effect on bacterial communities in meadow soils. The addition of biochar and nitrogen reduced the diversity of bacterial communities, and the more biochar was added, the diversity of bacterial communities was lower. Compared to the addition of raw biochar, the diversity of bacterial communities in small particle size biochar treatment was lower. This may be due to the fact that smaller particle size biochar exposes more sorption sites, under the condition of nitrogen source addition allows meadow soils to retain more ammonia nitrogen, which is toxic to many bacteria [45]. Nitrogen addition reduced the diversity of soil microbial bacterial communities, whereas the enrichment effect of biochar exacerbated this effect [46].

The species distribution map showed that single biochar addition without nitrogen addition in the fifth cycle of the experiment increased the relative abundance of Proteobacteria, Chloroflexi and Bacteroidota, and decreased the relative abundance of Acidobacteriota and Gemmatimonadota. Other studies also reported that biochar addition increased the relative abundance of Proteobacteria [47,48]. Chloroflexi can fix carbon dioxide in the presence of light [49], while biochar addition improved the permeability of the soil and facilitated this process. Bacteroides prefers nutrient-rich soils [50], and biochar enhances soil nutrients. Acidobacteria are suitable to grow in acidic environment, while biochar raises soil pH, therefore Acidobacteria mainly degrade polysaccharides in soil organic matter [51], which may also lead to a reduction in the efficiency of soil organic carbon mineralization. Gemmatimonadetes prefers to grow at higher temperatures and is strongly influenced by the inorganic phosphorus content in the soil [52]. The potential explanation for the increase in Proteobacteria abundance is that in our study, biochar increased soil TC, N, P and pH. This is consistent with Zhang et al.’s findings [53], where they reported a significant positive correlation between Proteobacterium and soil TC, N, P and pH. The functional flora analysis showed that biochar decreased the relative abundance of nitrifying and nitrite bacterial communities under the condition of urea addition, in the fifth cycle of the experiment, which may be due to that the increase in the relative abundance of nitrifying and nitrite bacterial communities in the short term because biochar addition increased the ammonia nitrogen content of the meadow soil [13]. In contrast, the enhanced nitrification in the long term led to a large consumption of the relevant substrates and a subsequent decrease in ammonia nitrogen content, while nitrate nitrogen accumulated in large amounts to inhibit nitrification, thus reducing the relative abundance of nitrifying and nitrite functional flora in the later stages. This study showed that biochar promoted biological nitrogen fixation by increasing the relative abundance of nitrogen-fixing bacteria. Analysis of significance of differences between groups showed that adding biochar significantly increased the relative abundance of Rhizobiaceae, Lysobacter and Xanthomonadales, as well as decreased the relative abundance of Cellulomonadaceae and Pedosphaerales. Both Rhizobiaceae and Xanthomonadales are aerobic bacteria and both have nitrogen fixation [54,55], which also indicated that biochar improved soil aeration and contributed to biological nitrogen fixation. Pedosphaerales mainly decompose polysaccharides [56], while Cellulomonadaceae decomposes cellulose [57], and the decrease in the relative abundance of these two species after adding biochar may also be one of the reasons accounting for the reduced effect of soil organic carbon mineralization.

KEGG metabolic pathway, COG function prediction and FAPROTAX microbial community function analysis were utilized the functional genes. In total, biochar addition increased gene abundance in nitrogen fixation and cell motility, while decreased gene abundance in nucleotide metabolism, carbohydrate metabolism, nitrate denitrification, nitrate respiration, nitrite denitrification and nitrite respiration. The results of functional gene analysis also indicated that biochar addition contributed to biological nitrogen fixation and inhibited microbial carbohydrate metabolism, which is one of the reasons why biochar addition increased soil carbon and nitrogen content.

5 Conclusions

The results showed that adding biochar to soils significantly increased the total organic carbon, ammonia nitrogen and total nitrogen contents. In addition, the enhancement of total organic carbon and total nitrogen content of meadow soil after biochar addition was higher than the carbon and nitrogen content of biochar itself, and the effect of small particle size biochar was better. The results of functional genetic analysis also indicated that biochar addition contributed to biological nitrogen fixation and inhibited microbial carbohydrate metabolism. This study also showed that biochar addition affected soil carbon and nitrogen content by influencing changes in bacterial community species and their abundance, including that biochar promoted biological nitrogen fixation by increasing the relative abundance of nitrogen-fixing bacteria. Our study found that biochar addition increased soil carbon and nitrogen content significantly, however, whether this effect still exists over a longer period of time, such as 5 or 10 years, and whether it still exists when crops are planted needs to be investigated in the future.

Author contribution

Pingnan Zhao: Conceptualization, Methodology, Data curation, Writing – original draft, Visualization, Investigation. Xiaoyuan Gao: Methodology, Data curation. Dong Liu: Visualization, Resources. Yuxuan Sun: Conceptualization，Data curation. Ming Li: Writing – review & editing. Song Han: Supervision, Funding acquisition.

Acknowledgments

The authors thank the supporters of this project and the referees for their constructive comments.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets are available from the corresponding author on reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/26395940.2023.2268272

Additional information

Funding

We are grateful for the grants from China Postdoctoral Science Foundation (2022M710647), Hei Long Jiang Postdoctoral Foundation (LBH-Z21085), the Fundamental Research Funds for the Central Universities (2572022BA09).